Spinal Nerve Ligation Injury Research Articles

CYP1B1-derived epoxides modulate the TRPA1 channel in chronic pain

Pain is often debilitating, and current treatments are neither universally efficacious nor without risks. Transient receptor potential (TRP) ion channels offer alternative targets for pain relief, but little is known about the regulation or identities of endogenous TRP ligands that affect inflammation and pain. Here, transcriptomic and targeted lipidomic analysis of damaged tissue from the mouse spinal nerve ligation (SNL)-induced chronic pain model revealed a time-dependent increase in Cyp1b1 mRNA and a concurrent accumulation of 8,9-epoxyeicosatrienoic acid (EET) and 19,20-EpDPA post injury. Production of 8,9-EET and 19,20-EpDPA by human/mouse CYP1B1 was confirmed invitro, and 8,9-EET and 19,20-EpDPA selectively and dose-dependently sensitized and activated TRPA1 in overexpressing HEK-293cells and Trpa1-expressing/AITC-responsive cultured mouse peptidergic dorsal root ganglia (DRG) neurons. TRPA1 activation by 8,9-EET and 19,20-EpDPA was attenuated by the antagonist A967079, and mouse TRPA1 was more responsive to 8,9-EET and 19,20-EpDPA than human TRPA1. This latter effect mapped to residues Y933, G939, and S921 of TRPA1. Intra-plantar injection of 19,20-EpDPA induced acute mechanical, but not thermal hypersensitivity in mice, which was also blocked by A967079. Similarly, Cyp1b1-knockout mice displayed a reduced chronic pain phenotype following SNL injury. These data suggest that manipulation of the CYP1B1-oxylipin-TRPA1 axis might have therapeutic benefit.

Chronicling changes in the somatosensory neurons after peripheral nerve injury

Current drug discovery efforts focus on identifying lead compounds acting on a molecular target associated with an established pathological state. Concerted molecular changes that occur in specific cell types during disease progression have generally not been identified. Here, we used constellation pharmacology to investigate rat dorsal root ganglion neurons using two models of peripheral nerve injury: chronic constriction injury (CCI) and spinal nerve ligation (SNL). In these well-established models of neuropathic pain, we show that the onset of chronic pain is accompanied by a dramatic, previously unreported increase in the number of bradykinin-responsive neurons, with larger increases observed after SNL relative to CCI. To define the neurons with altered expression, we charted the temporal course of molecular changes following 1, 3, 6, and 14 d after SNL injury and demonstrated that specific molecular changes have different time courses during the progression to a pain state. In particular, ATP receptors up-regulated on day 1 postinjury, whereas the increase in bradykinin receptors was gradual after day 3 postinjury. We specifically tracked changes in two subsets of neurons: peptidergic and nonpeptidergic nociceptors. Significant increases occurred in ATP responses in nAChR-expressing isolectin B4+ nonpeptidergic neurons 1 d postinjury, whereas peptidergic neurons did not display any significant change. We propose that remodeling of ion channels and receptors occurs in a concerted and cell-specific manner, resulting in the appearance of bradykinin-responsive neuronal subclasses that are relevant to chronic pain.

Peripheral nerve injury in rats induces alternations in choice behavior associated with food reinforcement.

Operant methods that allow animals to avoid painful stimuli are interpreted to assess the aversive quality of pain; however, such measurements require investigator-initiated stimuli to animals. Here we developed a shuttle maze test to repeatedly assess activity associated nociception without forced stimulation. Rats ambulate back and forth between two treat feeders by taking either a short route with a prickly surfaced arch or a longer route with a smooth floor. L5-L6 spinal nerve ligation (SNL) reduced the preference for the short route with the arch, correlated with hypersensitivity in the hind paw. Oral gabapentin restored the short route preference and reduced hypersensitivity in SNL rats, and blockade of spinal α2-adrenoceptors reduced gabapentin's effects on hypersensitivity but not on preference index. These results suggest that SNL injury alters behavior in the shuttle maze test and that the shuttle maze test shows comparable results to reflexive hypersensitivity after SNL in magnitude and response to gabapentin.

Differential Expression of Tubulin Acetylase and Deacetylase Between the Damaged Central and Peripheral Branch of Dorsal Root Ganglion Neurons.

BackgroundThe differences between the peripheral and central branches of the dorsal root ganglion (DRG) have not been fully elucidated. This study aimed to explore the expression of tubulin post-translational modifications (acetylation and deacetylation) between damaged peripheral and central branches of DRG neurons.Material/MethodsFifty Sprague-Dawley rats were randomly assigned to five groups with 10 rats in each group. These five groups consisted of spinal nerve ligation (SNL) at 24 hour and 48 hour, and cauda equina compression (CEC) at 24 hour and 48 hour, and a sham group. SNL injury in rats was induced by ligating L5 and L6 spinal nerves with 1-0 silk thread outboard the DRGs. CEC injury in rats was induced by a piece of silicone (10×1×1 mm) placed under the laminae of the L5–6 vertebra. Sham-operated rats underwent a simple laminectomy in L4, but silicone was not implanted. The expression profile of acetylase and deacetylase was examined by real-time PCR, Western blotting, and immunohistochemistry.ResultsIn the experimental groups, rats presented increased expression of acetylase (NAT1 and MEC-17) and decreased expression of deacetylase (Sirt2 and HDAC6) levels. Additionally, the expression of NAT1 and MEC-17 was gradually increased in DRG neurons following peripheral axonal injury compared to central axonal injury in a time-dependent manner. Conversely, the expression of Sirt2 and HDAC6 was gradually decreased in DRG neurons following peripheral axonal injury compared to central axonal injury in a time-dependent manner.ConclusionsOur study indicated that insufficiency of acetylase and upregulation of deacetylase in DRG neurons after central axonal injury may contribute to the pathogenesis of cauda equine syndrome.

Gabapentin loses efficacy over time after nerve injury in rats: role of glutamate transporter-1 in the locus coeruleus

Despite being one of the first-choice analgesics for chronic neuropathic pain, gabapentin sometimes fails to provide analgesia, but the mechanisms for this lack of efficacy is unclear. Rats with nerve injury including L5-L6 spinal nerve ligation (SNL) respond uniformly and well to gabapentin, but many of these studies are performed within just a few weeks of injury, questioning their relevance to chronic neuropathic pain. In this study, intraperitoneal gabapentin showed a time-dependently reduction in antihypersensitivity after SNL, associated with downregulation of astroglial glutamate transporter-1 (GLT-1) in the locus coeruleus (LC). Consistently, SNL also time-dependently increased basal but masked gabapentin-induced noradrenergic neuronal activity in the LC. In rats 2 weeks after SNL, knock-down of GLT-1 in the LC reduced the antihypersensitivity effect of gabapentin. In rats 8 weeks after SNL, increasing GLT-1 expression by histone deacetylase inhibitor valproate restored the antihypersensitivity effect of gabapentin, associated with restored gabapentin-induced noradrenergic neuronal activity in the LC and subsequent spinal noradrenaline release. Knock-down of GLT-1 in the LC reversed the effect of valproate to restore gabapentin-induced antihypersensitivity. In addition, the antihypersensitivity effect of the intrathecal α2-adrenoceptor agonist clonidine also decreased with time after SNL injury. These results suggest that downregulation of GLT-1 in the LC and reduced spinal noradrenergic inhibition contribute to impaired analgesic efficacy from gabapentin in chronic neuropathic pain and that valproate can rescue this impaired efficacy.

Analgesia for neuropathic pain by dorsal root ganglion transplantation of genetically engineered mesenchymal stem cells: initial results.

BackgroundCell-based therapy may hold promise for treatment of chronic pain. Mesenchymal stem cells (MSCs) are readily available and robust, and their secretion of therapeutic peptides can be enhanced by genetically engineering. We explored the analgesic potential of transplanting bone marrow-derived MSCs that have been transduced with lentivectors. To optimize efficacy and safety, primary sensory neurons were targeted by MSC injection into the dorsal root ganglia (DRGs).ResultsMSCs were transduced using lentivectors to express enhanced green fluorescent protein (EGFP) or to co-express the analgesic peptide glial cell line-derived neurotrophic factor (GDNF) and EGFP by a viral 2A bicistronic transgene cassette. Engineered MSCs were injected into the 4th lumbar (L4) and L5 DRGs of adult allogeneic rats to evaluate survival in the DRGs. MSCs were detected by immunofluorescence staining up to 2–3 weeks after injection, distributed in the extracellular matrix space without disrupting satellite glial cell apposition to sensory neurons, suggesting well-tolerated integration of engrafted MSCs into DRG tissue. To examine their potential for inhibiting development of neuropathic pain, MSCs were injected into the L4 and L5 DRGs ipsilateral to a spinal nerve ligation injury. Animals injected with GDNF-engineered MSCs showed moderate but significant reduction in mechanical allodynia and hyperalgesia compared to controls implanted with MSCs expressing EGFP alone. We also observed diminished long-term survival of allografted MSCs at 3 weeks, and the development of a highly-proliferating population of MSCs in 12% of DRGs after transplantation.ConclusionsThese data indicate that genetically modified MSCs secreting analgesic peptides could potentially be developed as a novel DRG-targeted cell therapy for treating neuropathic pain. However, further work is needed to address the challenges of MSC survival and excess proliferation, possibly with trials of autologous MSCs, evaluation of clonally selected populations of MSCs, and investigation of regulation of MSC proliferation.

A selective α2 B adrenoceptor agonist (A-1262543) and duloxetine modulate nociceptive neurones in the medial prefrontal cortex, but not in the spinal cord of neuropathic rats.

The noradrenergic system contributes to pain modulation, but the roles of its specific adrenoceptors are still being defined. We have identified a novel, potent (rat EC50 = 4.3 nM) and selective α2B receptor agonist, A-1262543, to further explore this adrenoceptor subtype's contribution to pathological nociception. Systemic administration of A-1262543 (1-10 mg/kg, intraperitoneal) dose-dependently attenuated mechanical allodynia in animals with a spinal nerve ligation injury. To further explore its mechanism of action, the activity of nociceptive neurones in the spinal cord and medial prefrontal cortex (mPFC) were examined after injection of 3 mg/kg of A-1262543 (intravenous, i.v.). These effects were compared with duloxetine (3 mg/kg, i.v.), a dual noradrenaline (NA) and serotonin (5-HT) reuptake inhibitor. Systemic administration of A-1262543 or duloxetine did not alter the spontaneous or evoked firing of spinal wide dynamic range and nociceptive-specific neurones in the neuropathic rats, indicating that neither compound engaged spinal, peripheral or descending pathways. In contrast to the lack of effect on spinal neurones, both A-1262543 and duloxetine reduced the evoked and spontaneous firing of 'pain-responsive' (PR) neurones in the mPFC. Duloxetine, but not A-1262543, also inhibited the firing of pain non-responsive (nPR) neurones in the mPFC probably reflecting duloxetine's contribution to modulating non-pain endpoints. These data highlight that activation of the α2B adrenoceptor as well as inhibiting NA and 5-HT reuptake can result in modulating the ascending nociceptive system, and in particular, dampening the firing of PR neurones in the mPFC.

Peripheral nerve injury reduces analgesic effects of systemic morphine via spinal 5-hydroxytryptamine 3 receptors.

Morphine produces powerful analgesic effects against acute pain, but it is not effective against neuropathic pain, and the mechanisms underlying this reduced efficacy remain unclear. Here, the authors compared the efficacy of systemic morphine between normal rats and rats with peripheral nerve injury, with a specific focus on descending serotonergic mechanisms. After L5 spinal nerve ligation injury, male Sprague-Dawley rats were subjected to behavioral testing, in vivo microdialysis of the spinal dorsal horn to determine serotonin (5-hydroxytryptamine [5-HT]) and noradrenaline release, and immunohistochemistry (n = 6 in each group). Intraperitoneal administration of morphine (1, 3, or 10 mg/kg) produced analgesic effects in normal and spinal nerve ligation rats, but the effects were greater in normal rats (P < 0.001). Morphine increased 5-HT release (450 to 500% of the baseline), but not noradrenaline release, in the spinal dorsal horn via activation of serotonergic neurons in the rostral ventromedial medulla. Intrathecal pretreatment with ondansetron (3 μg), a 5-HT3 receptor antagonist, or 5,7-dihydroxytryptamine creatinine sulfate (100 μg), a selective neurotoxin for serotonergic terminals, attenuated the analgesic effect of morphine (10 mg/kg) in normal rats but increased the analgesic effect of morphine in spinal nerve ligation rats (both P < 0.05). Systemic administration of morphine increases 5-HT levels in the spinal cord, and the increase in 5-HT contributes to morphine-induced analgesia in the normal state but attenuates that in neuropathic pain through spinal 5-HT3 receptors. The plasticity of the descending serotonergic system may contribute to the reduced efficacy of systemic morphine in neuropathic pain.

Reversal of Peripheral Nerve Injury-induced Hypersensitivity in the Postpartum Period

Physical injury, including surgery, can result in chronic pain; yet chronic pain following childbirth, including cesarean delivery in women, is rare. The mechanisms involved in this protection by pregnancy or delivery have not been explored. We examined the effect of pregnancy and delivery on hypersensitivity to mechanical stimuli of the rat hindpaw induced by peripheral nerve injury (spinal nerve ligation) and after intrathecal oxytocin, atosiban, and naloxone. Additionally, oxytocin concentration in lumbar spinal cerebrospinal fluid was determined. Spinal nerve ligation performed at mid-pregnancy resulted in similar hypersensitivity to nonpregnant controls, but hypersensitivity partially resolved beginning after delivery. Removal of pups after delivery prevented this partial resolution. Cerebrospinal fluid concentrations of oxytocin were greater in normal postpartum rats prior to weaning. To examine the effect of injury at the time of delivery rather than during pregnancy, spinal nerve ligation was performed within 24 h of delivery. This resulted in acute hypersensitivity that partially resolved over the next 2-3 weeks. Weaning of pups resulted only in a temporary return of hypersensitivity. Intrathecal oxytocin effectively reversed the hypersensitivity following separation of the pups. Postpartum resolution of hypersensitivity was transiently abolished by intrathecal injection of the oxytocin receptor antagonist, atosiban. These results suggest that the postpartum period rather than pregnancy protects against chronic hypersensitivity from peripheral nerve injury and that this protection may reflect sustained oxytocin signaling in the central nervous system during this period.

Effects of Ethyl Pyruvate on Allodynia, TNF-α Expression, and Apoptosis in the Dorsal Root Ganglion after Spinal Nerve Ligation Injury

BackgroundIt has been demonstrated that the expression of tumor necrosis factor-α (TNF-α) and apoptotic cell death in the dorsal root ganglion (DRG) following spinal nerve constriction injury play a role in the initiation and continuation of hyperalgesia and allodynia. The present study was designed to investigate the effects of ethyl pyruvate (EP) on mechanical and cold allodynia, TNF-α expression, and apoptosis in DRG after spinal nerve ligation injury.MethodsRats were divided into 3 groups: control, pre-EP, and post-EP. EP (50 mg/kg) was intraperitoneally injected 30 minutes before (pre-EP) or after (post-EP) surgery. Behavioral tests to determine mechanical and cold allodynia were conducted before surgery and 4 and 7 days after surgery. Seven days after surgery, TNF-α protein levels in DRG were evaluated by enzyme-linked immunosorbent assay, and DRG apoptosis was determined by immunohistochemical detection of activated caspase-3.ResultsTreatment with EP significantly reduced mechanical and cold allodynia following spinal nerve ligation injury. TNF-α protein levels in the pre-EP (4.7 ± 1.2 pg/200 µg; P < 0.001) and post-EP (6.4 ± 1.8 pg/200 µg; P < 0.001) groups were 2-3 times lower than the control group (14.4 ± 1.2 pg/200 µg). The percentages of neurons and satellite cells that co-localized with caspase-3 were also significantly lower in the pre-EP and post-EP groups than the control group.ConclusionsThese results demonstrate that EP has a strong anti-allodynic effect that acts through the inhibition of TNF-α expression and apoptosis in DRG after spinal nerve ligation injury.

Thrombospondin-4 Contributes to Spinal Sensitization and Neuropathic Pain States

Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-gene chip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naive rats is sufficient to enhance the frequency of EPSCs in spinal dorsal horn neurons, suggesting an increased excitatory presynaptic input, and to cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal presynaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.

Tumor Necrosis Factor-alpha and Apoptosis Following Spinal Nerve Ligation Injury in Rats

BackgroundSpinal nerve ligation (SNL) injury in rats produces a pain syndrome that includes mechanical and thermal allodynia. Previous studies have indicated that proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) play an important role in peripheral mediation of neuropathic pain, and that altered dorsal root ganglion (DRG) function and degree of DRG neuronal apoptosis are associated with spinal nerve injury. The present study was conducted to evaluate the expression of TNF-α and the extent of apoptosis in the dorsal root ganglion after SNL in rats.MethodsSprague-Dawley rats were subjected to SNL of the left L5 and L6 spinal nerves distal to the DRG and proximal to the formation of the sciatic nerve. At postoperative day 8, TNF-α protein levels in the L5-6 DRG were compared between SNL and naive groups using ELISA. In addition, we compared the percentage of neurons injured in the DRG using immunostaining for apoptosis and localization of activated caspase-3.ResultsSNL injury produced significant mechanical and cold allodynia throughout the 7-day experimental period. TNF-α protein levels were increased in the DRG in rats that had undergone SNL (12.7 ± 3.2 pg/100 µg, P < 0.001) when compared with naïve rats (4.1 ± 1.4 pg/100 µg). The percentage of neurons or satellite cells co-localized with activated caspase-3 were also significantly higher in rats with SNL than in naïve rats (P < 0.001, P < 0.05, respectively).ConclusionsSNL injury produces mechanical and cold allodynia, as well as TNF-α elevation and apoptosis in the DRG.

Manual acupuncture inhibits mechanical hypersensitivity induced by spinal nerve ligation in rats

Manual acupuncture (MA) has presented analgesic activity against neuropathic pain in patients and animal models, yet a series of questions remain: Is MA effectiveness dependent of acupoint selection or combination? Is it equally efficient when treatment starts on the initial (acute) or sub-chronic phase of spinal nerve ligation (SNL)-induced neuropathy? Is MA effect related to the release of endogenous opioids? Does MA produce similar effects to gabapentin? To answer these questions rats submitted to the L5/L6 SNL injury were treated with unilateral MA (ST36 (Zusanli), SP6 (Sanyingjiao) or ST36+SP6 acupoint stimulation); or with gabapentin (30 mg/kg i.p., used as positive control). Both acupoints have been demonstrated to present analgesic activity and are used in clinical practice and basic science research. In addition, we investigated the influence of naloxone (1 mg/kg i.p., a nonselective opioid receptor antagonist) on MA treatment and also the effect of unilateral ST36+SP6 MA treatment beginning acutely (5 days) or sub-chronically (14 days) after SNL. Our results demonstrate that single or combined unilateral stimulation was able to reduce mechanical hypersensitivity with treatment beginning in both acute and sub-chronic phases of SNL-induced neuropathy; MA effect was blocked by naloxone, and finally; SP6+ST36 MA presented similar effect to gabapentin (30 mg/kg). In conclusion, our results demonstrate, for the first time, that unilateral MA (ST36, SP6 or ST36+SP6) reduces hypersensitivity induced by the SNL with effect dependent of the opioid system and comparable with the one obtained with gabapentin (used as positive control).

Profiling of dynamically changed gene expression in dorsal root ganglia post peripheral nerve injury and a critical role of injury-induced glial fibrillary acetic protein in maintenance of pain behaviors

To explore cellular changes in sensory neurons after nerve injury and to identify potential target genes contributing to different stages of neuropathic pain development, we used Affymetrix oligo arrays to profile gene expression patterns in L5/6 dorsal root ganglia (DRG) from the neuropathic pain model of left L5/6 spinal nerve ligation at different stages of neuropathic pain development. Our data indicated that nerve injury induced changes in expression of genes with similar biological functions in a temporal specific manner that correlates with particular stages of neuropathic pain development, indicating dynamic neuroplasticity in the DRG in response to peripheral nerve injury and during neuropathic pain development. Data from post-array validation indicated that there was a temporal correlation between injury-induced expression of the glial fibrillary acidic protein (GFAP), a marker for activated astrocytes, and neuropathic pain development. Spinal nerve ligation injury in GFAP knockout mice resulted in neuropathic pain states with similar onset, but a shortened duration compared with that in age, and gender-matched wild-type littermates. Intrathecal GFAP antisense oligonucleotide treatment in injured rats with neuropathic pain states reversed injury-induced behavioral hypersensitivity and GFAP upregulation in DRG and spinal cord. Together, these findings indicate that injury-induced GFAP upregulation not only serves as a marker for astrocyte activation, but it may also play a critical, but yet identified, role in the maintenance of neuropathic pain states.

Enhanced Pre-Synaptic Glutamate Release in Deep-Dorsal Horn Contributes to Calcium Channel Alpha-2-Delta-1 Protein-Mediated Spinal Sensitization and Behavioral Hypersensitivity

Nerve injury-induced expression of the spinal calcium channel alpha-2-delta-1 subunit (Cavα2δ1) has been shown to mediate behavioral hypersensitivity through a yet identified mechanism. We examined if this neuroplasticity modulates behavioral hypersensitivity by regulating spinal glutamatergic neurotransmission in injury-free transgenic mice overexpressing the Cavα2δ1 proteins in neuronal tissues. The transgenic mice exhibited hypersensitivity to mechanical stimulation (allodynia) similar to the spinal nerve ligation injury model. Intrathecally delivered antagonists for N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptors, but not for the metabotropic glutamate receptors, caused a dose-dependent allodynia reversal in the transgenic mice without changing the behavioral sensitivity in wild-type mice. This suggests that elevated spinal Cavα2δ1 mediates allodynia through a pathway involving activation of selective glutamate receptors. To determine if this is mediated by enhanced spinal neuronal excitability or pre-synaptic glutamate release in deep-dorsal horn, we examined wide-dynamic-range (WDR) neuron excitability with extracellular recording and glutamate-mediated excitatory postsynaptic currents with whole-cell patch recording in deep-dorsal horn of the Cavα2δ1 transgenic mice. Our data indicated that overexpression of Cavα2δ1 in neuronal tissues led to increased frequency, but not amplitude, of miniature excitatory post synaptic currents mediated mainly by AMPA/kainate receptors at physiological membrane potentials, and also by NMDA receptors upon depolarization, without changing the excitability of WDR neurons to high intensity stimulation. Together, these findings support a mechanism of Cavα2δ1-mediated spinal sensitization in which elevated Cavα2δ1 causes increased pre-synaptic glutamate release that leads to reduced excitation thresholds of post-synaptic dorsal horn neurons to innocuous stimuli. This spinal sensitization mechanism may mediate at least partially the neuropathic pain states derived from increased pre-synaptic Cavα2δ1 expression.

Oxycodone Plus Ultra-Low-Dose Naltrexone Attenuates Neuropathic Pain and Associated μ-Opioid Receptor–Gs Coupling

Both peripheral nerve injury and chronic opioid treatment can result in hyperalgesia associated with enhanced excitatory neurotransmission at the level of the spinal cord. Chronic opioid administration leads to a shift in mu-opioid receptor (MOR)-G protein coupling from G(i/o) to G(s) that can be prevented by cotreatment with an ultra-low-dose opioid antagonist. In this study, using lumbar spinal cord tissue from rats with L(5)/L(6) spinal nerve ligation (SNL), we demonstrated that SNL injury induces MOR linkage to G(s) in the damaged (ipsilateral) spinal dorsal horn. This MOR-G(s) coupling occurred without changing G(i/o) coupling levels and without changing the expression of MOR or Galpha proteins. Repeated administration of oxycodone alone or in combination with ultra-low-dose naltrexone (NTX) was assessed on the SNL-induced MOR-G(s) coupling as well as on neuropathic pain behavior. Repeated spinal oxycodone exacerbated the SNL-induced MOR-G(s) coupling, whereas ultra-low-dose NTX cotreatment slightly but significantly attenuated this G(s) coupling. Either spinal or oral administration of oxycodone plus ultra-low-dose NTX markedly enhanced the reductions in allodynia and thermal hyperalgesia produced by oxycodone alone and minimized tolerance to these effects. The MOR-G(s) coupling observed in response to SNL may in part contribute to the excitatory neurotransmission in spinal dorsal horn in neuropathic pain states. The antihyperalgesic and antiallodynic effects of oxycodone plus ultra-low-dose NTX (Oxytrex, Pain Therapeutics, Inc., San Mateo, CA) suggest a promising new treatment for neuropathic pain. The current study investigates whether Oxytrex (oxycodone with an ultra-low dose of naltrexone) alleviates mechanical and thermal hypersensitivities in an animal model of neuropathic pain over a period of 7 days, given locally or systemically. In this report, we first describe an injury-induced shift in mu-opioid receptor coupling from G(i/o) to G(s), suggesting why a mu-opioid agonist may have reduced efficacy in the nerve-injured state. These data present a novel approach to neuropathic pain therapy.

Proteomics study of neuropathic and nonneuropathic dorsal root ganglia: altered protein regulation following segmental spinal nerve ligation injury

Peripheral nerve injury is often followed by the development of severe neuropathic pain. Nerve degeneration accompanied by inflammatory mediators is thought to play a role in generation of neuropathic pain. Neuronal cell death follows axonal degeneration, devastating a vast number of molecules in injured neurons and the neighboring cells. Because we have little understanding of the cellular and molecular mechanisms underlying neuronal cell death triggered by nerve injury, we conducted a proteomics study of rat 4th and 5th lumbar (L4 and L5) dorsal root ganglion (DRG) after L5 spinal nerve ligation. DRG proteins were displayed on two-dimensional gels and analyzed through quantitative densitometry, statistical validation of the quantitative data, and peptide mass fingerprinting for protein identification. Among approximately 1,300 protein spots detected on each gel, we discovered 67 proteins that were tightly regulated by nerve ligation. We find that the injury to primary sensory neurons turned on multiple cellular mechanisms critical for the structural and functional integrity of neurons and for the defense against oxidative damage. Our data indicate that the regulation of metabolic enzymes was carefully orchestrated to meet the altered energy requirement of the DRG cells. Our data also demonstrate that ligation of the L5 spinal nerve led to the upregulation in the L4 DRG of the proteins that are highly expressed in embryonic sensory neurons. To understand the molecular mechanisms underlying neuropathic pain, we need to comprehend such dynamic aspect of protein modulations that follow nerve injury.

Descending facilitation from the rostral ventromedial medulla maintains nerve injury-induced central sensitization

Nerve injury can produce hypersensitivity to noxious and normally innocuous stimulation. Injury-induced central (i.e. spinal) sensitization is thought to arise from enhanced afferent input to the spinal cord and to be critical for expression of behavioral hypersensitivity. Descending facilitatory influences from the rostral ventromedial medulla have been suggested to also be critical for the maintenance, though not the initiation, of experimental neuropathic pain. The possibility that descending facilitation from the rostral ventromedial medulla is required for the maintenance of central sensitization was examined by determining whether ablation of mu-opioid receptor-expressing cells within the rostral ventromedial medulla prevented the enhanced expression of repetitive touch-evoked FOS within the spinal cord of animals with spinal nerve ligation injury as well as nerve injury-induced behavioral hypersensitivity. Rats received a single microinjection of vehicle, saporin, dermorphin or dermorphin–saporin into the rostral ventromedial medulla and 28 days later, underwent either sham or spinal nerve ligation procedures. Animals receiving rostral ventromedial medulla pretreatment with vehicle, dermorphin or saporin that were subjected to spinal nerve ligation demonstrated both thermal and tactile hypersensitivity, and showed significantly increased expression of touch-evoked FOS in the dorsal horn ipsilateral to nerve injury compared with sham-operated controls at days 3, 5 or 10 post-spinal nerve ligation. In contrast, nerve-injured animals pretreated with dermorphin–saporin showed enhanced behaviors and touch-evoked FOS expression in the spinal dorsal horn at day 3, but not days 5 and 10, post-spinal nerve ligation when compared with sham-operated controls. These results indicate the presence of nerve injury-induced behavioral hypersensitivity associated with nerve injury-induced central sensitization. Further, the results demonstrate the novel concept that once initiated, maintenance of nerve injury-induced central sensitization in the spinal dorsal horn requires descending pain facilitation mechanisms arising from the rostral ventromedial medulla.

Differential blockade of nerve injury–induced thermal and tactile hypersensitivity by systemically administered brain-penetrating and peripherally restricted local anesthetics

Systemic administration of local anesthetics has been shown to transiently reverse thermal and tactile hypersensitivity induced by peripheral nerve injury, effects that have been taken as suggesting direct actions on the peripheral nerves. The present study sought to determine whether a central site of action could contribute to, or account for, the effects of lidocaine on nerve injury–induced thermal and tactile hypersensitivity. Systemic lidocaine and its peripherally restricted analogues, QX-314 or QX-222, effectively reversed thermal hypersensitivity after spinal nerve ligation injury. Nerve injury–induced tactile hypersensitivity, however, was reversed by systemic lidocaine but not QX-314 or QX-222. Microinjection of either lidocaine or QX-314 into the rostral ventromedial medulla fully reversed spinal nerve ligation–induced thermal and tactile hypersensitivity. The data strongly suggest that nerve injury–induced thermal and tactile hypersensitivity are mediated through different mechanisms. In addition, the present study supports a prominent contribution of the central nervous system in the activity of systemically given lidocaine against nerve injury–induced tactile and thermal hypersensitivity. Thus, lidocaine might reverse tactile hypersensitivity mainly through its actions within the central nervous system, whereas its reversal of thermal hypersensitivity might occur through either central or peripheral sites. Perspective Nerve injury–induced neuropathic pain has proved remarkably difficult to treat. Systemic administration of ion channel blockers such as lidocaine has been explored for the management of chronic pain. This work indicates that systemic rather than local administration of lidocaine would be more effective in treating tactile allodynia.

The Effect of Intradermal Norepinephrine on Allodynia in a Neuropathic Pain Rat Model

Background: A spinal nerve ligation (SNL) injury may produce a neuropathic pain syndrome that includes tactile allodynia. This pain state may be diminished by sympathectomy. Intradermal (I.D.) injection of norepinephrine (NE) evokes pain in patients with sympathetically maintained pain. Recently, we reported the effect of intrathecal (I.T.) brimonidine and rilmenidine on the sympathetic nervous system. Therefore, we conducted a behavioral test to investigate the effects of sympathetic stimulation by I.D. NE on mechanical allodynia in rats with a SNL injury. Methods: Male SD rats were prepared with ligation of the left lumbar 5th and 6th spinal nerves and lumbar I.T. catheter implantation. NE 10g I.D. was administered in normal and SNL rats to investigate the change of cutaneous sensitivity to tactile stimuli. NE 30g I.D. was administered before and after I.T. injection of brimonidine 3g and rilmenidine 30g in SNL rats. Using a von Frey hair (VFH) test, we examined the effects of NE on the withdrawal threshold. Allodynic thresholds for the withdrawal response of the lesioned hindpaw to VFH stimuli were assessed. Results: Intradermal NE produced a reduction of the withdrawal threshold in normal and allodynic rats. An allodynic state induced by a SNL was aggravated by NE. In allodynic rats, the baseline threshold of a lesioned left hindpaw was markedly low and such a state was maintained during the behavioral experiment. The antiallodynic effects of I.T. brimonidine and rilmenidine were produced in both pre- and post-treatment of NE. Conclusions: The results suggest that a sympathetic component is likely involved in the mechanism of mechanical allodynia produced by a SNL injury.